Biomedical Engineering and Bioengineering Commons^™

Modification Reactivity Analysis Of Human Replication Protein A In Biologically Important States, Ryan James Yoakum

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Human Replication Protein A (RPA) is a heterotrimeric protein consisting of 70, 32, and 14 kDa subunits. RPA is the predominant single stranded DNA binding protein within the cell. It is involved in all forms of the DNA metabolic pathways, including but not limited to, replication, recombination, damage repair, as well as cell cycle and DNA check point signaling. RPA is phosphorylated (pRPA) during G1-S phase and is dephosphorylated during M phase. Further, RPA is hyperphosphorylated during DNA damage. Through the use of x-ray crystallography and nuclear magnetic resonance, researchers have proposed models and structures based on truncated portions of …

A Computational Tool For Biomolecular Structure Analysis Based On Chemical And Enzymatic Modification Of Native Proteins, Deacon John Sweeney

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Chemical and enzymatic modification of proteins is a well-established treatment technique for probing the conformational properties of these macromolecules. Investigators have recently extended the approach to probe many sites on a protein's structure in parallel manner, such that conformational properties of a target protein can be inferred. The modern approach uses mass spectrometry to quantify reactant loss and product formation. Rigorous analysis is challenging due to the high volume of mass spectrometric data that must be processed and interpreted.

An extensively interactive software suite has been developed to assist various aspects of the analytical protocol. The software offers a variety …

Novel Redox And Dna-Dependent Conformational Changes In Human Ku, A Dna-Double Strand Break Repair Protein, Jason Alexander Lehman

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Ionizing radiation (IR) and radiomimetic drugs used in cancer chemotherapy cause DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. Ku is a heterodimeric protein comprised of 70 and 80 kDa subunits and recognizes free DNA ends. Once Ku is bound to DNA, it binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and forms a heterotrimeric DNA-PK complex for repair and damage signaling.

We have analyzed the Ku protein using multiple biochemical techniques and uncovered a novel reversible redox change (Andrews, Lehman and Turchi, (2006) JBC 281(19):13596-603). From this data, we hypothesized that a redox-dependent …

Investigating The Role Of Subunit Iii In The Structure And Function Of Rhodobacter Sphaeroides Cytochrome C Oxidase, R. Ryan Geyer

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The role of subunit III (SIII) in cytochrome c oxidase structure and function was investigated using enzyme isolated from the bacterium Rhodobacter sphaeroides. Energy minimization calculations suggested that in the absence of SIII, subunit I (SI) adopted a more open conformation. This observation was tested through the use of limited proteolysis using, á-chymotrypsin. The results showed that in the absence of SIII the solution structures of wild-type and I/II oxidase were not significantly different, and that proteolysis occurred exclusively at the N and C-termini of SI. Upon inactivation of I/II oxidase by catalytic turnover, and subsequent digestion with the protease …